ALKBH1 was recently discovered as a demethylase for DNA N6-methyladenine (N6-mA), a new epigenetic modification, and interacts with the core transcriptional pluripotency network of embryonic stem cells. However, the role of ALKBH1 and DNA N6-mA in regulating osteogenic differentiation is largely unknown. In this study, we demonstrated that the expression of ALKBH1 in human mesenchymal stem cells (MSCs) was upregulated during osteogenic induction. Knockdown of ALKBH1 increased the genomic DNA N6-mA levels and significantly reduced the expression of osteogenic-related genes, alkaline phosphatase activity, and mineralization. ALKBH1-depleted MSCs also exhibited a restricted capacity for bone formation in vivo. By contrast, the ectopic overexpression of ALKBH1 enhanced osteoblastic differentiation. Mechanically, we found that the depletion of ALKBH1 resulted in the accumulation of N6-mA on the promoter region of ATF4, which subsequently silenced ATF4 transcription. In addition, restoring the expression of ATP by adenovirus-mediated transduction successfully rescued osteogenic differentiation. Taken together, our results demonstrate that ALKBH1 is indispensable for the osteogenic differentiation of MSCs and indicate that DNA N6-mA modifications area new mechanism for the epigenetic regulation of stem cell differentiation.Human bone marrow-derived MSCs were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) plus 100 U·mL−1 of penicillin and 100 mg·mL−1 of streptomycin (Gibco) at 37 °C with a humidified atmosphere of 5% CO2. To induce osteogenic differentiation, MSCs were seeded in 6- or 24-well plates. After confluence, cells were treated with osteogenic medium containing 50 μmol·L−1 ascorbic acid, 10 mmol·L−1 β-glycerophosphate, and 10 nmol·L−1 dexamethasone (Sigma, Shanghai, China). All experimental protocols and procedures were approved by the State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University.We first evaluated the expression profile of ALKBH1 in human MSCs during osteogenic differentiation. As determined by real-time RT-PCR, the ALKBH1 messenger RNA levels were significantly upregulated in response to osteogenic induction (Figure 1a). This observation was also confirmed by western blot analysis (Figure 1b). These results suggest that ALKBH1 may have a role in the osteogenic differentiation of MSCs.This work was supported by grants from the National Natural Science Foundation of China (No. 81271178 and 81470777).